DNA ladders are common reagents in molecular biology, useful for determining the size of DNA fragments. A DNA ladder comprises two or more DNA fragments of known size. Typically, a DNA sample and a DNA ladder are loaded in adjacent wells of an agarose gel. The DNA is separated by electrophoresis through the gel. The gel is stained with a flourescent dye, such as ethidium bromide, and exposed to ultraviolet light. The size of the sample DNA fragments are determined by comparing their migration with the bands of known size in the DNA ladder. DNA ladders are commercially available from numerous vendors, including Sigma, Pharmacia, Life Technologies, Promega, Boerhinger-Mannheim, Amersham, New England Biolabs, Stratagene, and Invitrogen.
One established method for manufacturing a DNA ladder is by partial restriction digestion of a special plasmid. The special plasmid contains an insert of tandem repeats of a DNA fragment. The same unique restriction site lies at each junction of the repeat units. Partial restriction digestion of this plasmid produces a ladder containing multimers of the repeated DNA fragment. For example, assume that a special plasmid contains an insert with the following characteristics: (i) the insert consists of a ten tandem repeat of a 100 base pair (bp) DNA fragment, (ii) the tandem repeat units are joined to each other and to the cloning vector by AvaI restriction sites. Partial restriction digestion with Aval would produce a DNA ladder with fragments from 100 bp to 1,000 bp in 100 bp increments.
Constructing special plasmids of this type is established in the art. Such a plasmid is constructed by cloning multiple monomer DNA fragments in tandem into a cloning vector. Typically, the vector is pUC18, which is 2,686 bp. In the tandem repeat, the monomer fragments are usually oriented in a head to tail manner by using the cloning method of Hartley (U.S. Pat. No. 4,403,036). In Hartley's method, the monomer DNA fragment contains Aval sites at each end which force polymerization to occur in a head to tail manner. The head to tail orientation improves plasmid stability, but it is not essential for the method of ladder production.
There are three problems with the established approach.
(1) Only the portion of the plasmid containing the tandem repeat can generate the ladder. The vector portion of the plasmid is not used to generate the ladder. PA1 (2) The size of the largest DNA fragment in the ladder is limited to the size of the tandem repeat region. PA1 (3) Restriction fragments containing the vector region of the plasmid produce a large smear of by-product fragments in the upper portion of the ladder. This upper smear does not contribute useful bands to the ladder.
Thus, it is well established in the art that ladders can be produced by partial restriction digestion of plasmids which contain tandem repeats of a DNA fragment. It is also well established that such ladders suffer three drawbacks: (1) Only the tandem repeat portion of the plasmid generates the ladder, (2) the largest DNA fragment is limited to the size of the tandem repeat, and (3) the upper bands containing the vector region of the plasmid do not contribute useful bands to the ladder. In this context, a useful approach to solve all these problems has been devised by the inventor.